Cell cultures were established from a benign pancreatic islet adenoma. Over 200 muU/culture/day immunoreactive insulin were found in culture media. Cultures with medium 199 released insulin for about 2 months; those with medium F12K were maintained for over 7 months, and have been successfully subcultured. Increasing culture medium glucose to 326 mg per 100 ml, alone or with leucine (10 mM) or theophylline (2 mM), failed to increase insulin release above baseline. Studies in the patient prior to surgery using oral glucose, leucine, beef meal, intravenous tolbutamide, and glucagon failed to increase plasma insulin and thus were consistent with cell culture responses. Extracts of tumor tissue contained 23% proinsulin-like material; high insulin containing samples of culture medium had 5% proinsulin and less than 40 pg glucagon/ml. Aldehyde fuchsin positive granulation was sparse in both cultured cells and the original tumor. These studies demonstrate long term viability, in monolayer culture, of cells derived from this islet cell adenoma, with retention of secretory characteristics consistent with data obtained prior to removal of the adenoma from the patient.